The expression of cloned or purposefully altered genes in transgenic organisms has been seen to hold great promise for the production of substances by transgenic "bioreactors" and for the production of improved animals and plants, among other applications. Obstacles to realizing the promise have been encountered, however. In particular, it has not been possible to obtain highly efficient production of properly modified useful substances in transgenic animals. The present invention relates to methods and transgenic organisms that overcome the problem and to products produced by the transgenic organisms, inter alia.
Many DNAs have been cloned and expressed in cells in culture to produce heterologous proteins, peptides and other substances. Several genes also have been introduced into plants or animals to produce heterologous proteins, peptides and other substances. Although it has been possible, in general, to engender expression and production of proteins and other substances in cells, animals and plants by expressing cloned genes, the production levels that have been obtained often have been low and the posttranslational processing of proteins produced this way generally has been incomplete or inefficient. These difficulties particularly have limited, or precluded, the use of animal and plant bioreactors to produce proteins with the proper posttranslational modifications. Low production levels and low specific activities in cultured cells have been attributed to the expression of limiting amounts of particular enzymes necessary for the production of the expressed protein in its properly modified form.
Attempts have been made to increase production of properly modified proteins in cells in culture by expressing a cloned gene to increase the amount of a limiting enzyme activity in the cells. For instance, expression of a transfected yeast Kex2 cDNA in baby hamster kidney ("BHK") cells that expressed human Protein C ("HPC") from an amplified Protein C gene increased the conversion of Protein C from the single-chain zymogen form to the mature two-chain form (Foster et al., Biochemistry 30: 367-372 (1991).
In another example, PACE/furin expressed at high levels by a transfected DNA in chinese hamster ovary cells ("CHO") apparently increased proper cleavage of the propeptide of the co-expressed Factor IX precursor (Wasley et al., J. Biol. Chem. 268: 8458-8465 (1993)).
But, the processing activity apparently engendered by expression of the PACE/furin gene in these experiments was difficult to discern and varied. In addition, apparent increases in protease activity seemed to cause cell toxicity, cytopathic effect and alterations in markers of cellular differentiation. Indeed, it has been suggested that PACE/furin and similar processing enzymes may be deleterious or lethal to cells when they are inappropriately expressed, even in culture, as noted for instance by Schalken et al., J. Clin. Invest. 80: 1545-1549 (1987), Ayoubi et al., J. Biol. Chem. 269: 9298-9303 (1994) and Decroly et al., J. Biol. Chem. 269: 12240-12247 (1994). The potential for improving the cellular production of substances in this way is overshadowed by the adverse affects observed in culture.
In light of such results, this approach to improving production of proteins, polypeptides and other substances in transgenic organisms has not been favorably considered. For one, expression of enzymes that alter posttranslational modification in cells thus far has been carried out only in abnormal cultured cells. These cells generally exhibit aberrant growth, which allows them to propagate indefinitely in artificial media. Largely, such cells are derived from tumors or are the outcome of transduction with immortalizing viruses. Differentiation and growth factors particularly are altered in such cells. Thus, the response of these cells to altered posttranslational modification capacity does not indicate the expected response of cells in a healthy organism.
Indeed, it has been thought that such cells are far more tolerant than an intact organism of adverse effects stemming from altered expression of enzymes affecting posttranslational modifications. Given the role of posttranslational modifications in controlling enzyme cascades and in modulating the activity of factors that control growth, mitosis and differentiation, including processes that generally occur only in intact organisms, altering posttranslational modification capacities poses a greater risk of being severely deleterious to an intact organism, even when it would not adversely impact a cell grown in culture.
A developing organism is especially sensitive to inappropriate expression, particularly expression that causes cytopathic effect. For example, loss of a single cell at a critical stage can hopelessly incapacitate or abort a developing embryo.
In addition, an organism can be adversely affected not only by intracellular effects but also by physiological communication of the activity from expressing cells to other parts of the organism. The complex physiological processes involved in transport and metabolism of circulating proteins, moreover, often has the potential to amplify a tolerable intracellular effect into an effect that is intolerably damaging to the organism as a whole.
For instance, it has been noted that expression of some enzymes that carry out posttranslational modifications can activate toxins. Altering a posttranslational modification that activates a toxin thus, potentially could amplify ordinarily tolerable toxin levels, such as those produced by a mild infection, to levels seriously harmful or lethal to an organism (Chiron et al., J. Biol. Chem. 269: 18167-18176 (1994)).
A variety of enzymes involved in posttranslational modification of proteins, moreover, are expressed differently in the cells of different tissues in an organism. In addition, expression of such proteins in cells often varies greatly over the course of embryonic development. This is the case, for instance, of the subtilisin-like PACEs, such as PACE/furin, which appear to carry out proteolytic steps in the maturation of some proteins. In fact, it has been suggested that posttranslational modifications, such as the proteolytic cleavages mediated by these processing proteases, not only participate in, but also have a regulatory role in the process of cell differentiation (Zheng et al., J. Neuroscience, 14: 4656-4673 (1994). Inappropriate expression of these enzymes in cells of a developing embryo thus could have broadly deleterious or lethal effects.
Concern about deleterious effects in vivo have made it seem unlikely that posttranslational modifications of proteins can be much altered in cells of transgenic organisms. Thus, the idea of changing the posttranslational modification properties of cells has not seemed to be a promising way to improve the ability of transgenic organisms to produce useful substances. Given the potential of such animals for the production of useful substances, it therefore remains an important goal to devise methods that overcome these obstacles to developing efficient bioreactors.